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WO1993003378A1 - Procede de depistage de cellules tumorales metastatiques - Google Patents

Procede de depistage de cellules tumorales metastatiques Download PDF

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Publication number
WO1993003378A1
WO1993003378A1 PCT/US1992/006237 US9206237W WO9303378A1 WO 1993003378 A1 WO1993003378 A1 WO 1993003378A1 US 9206237 W US9206237 W US 9206237W WO 9303378 A1 WO9303378 A1 WO 9303378A1
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hla
tumor cells
cells
human
protein
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PCT/US1992/006237
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English (en)
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Mohanathasan Chelladurai
Kenneth V. Honn
Daniel A. Walz
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Radiation Oncology Center Research And Development Corporation
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Publication of WO1993003378A1 publication Critical patent/WO1993003378A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
    • G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2474/00—Immunochemical assays or immunoassays characterised by detection mode or means of detection
    • G01N2474/20—Immunohistochemistry assay
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/811—Test for named disease, body condition or organ function
    • Y10S436/813—Cancer

Definitions

  • the present invention relates to a method for testing for the presence of metastatic human tumor cells by determining the level of Human Leukocyte Antigen (HLA) in the tumor cells.
  • HLA Human Leukocyte Antigen
  • the present invention relates to a method which uses a bacterial toxin or subunit thereof which selectively binds the HLA to provide a basis for the detection and level of activity.
  • Trousseau observed a high incidence of venous thrombosis in patients with gastric carcinoma and described the syndrome which bears his name (Trousseau, A., In Clinigue Medicale de 1 'Hotel-Dieu de Paris; Paris, Balliere, 3:654 (1865)).
  • a considerable body of evidence supports an association between cancer and thromboembolic disorders (Rickles, F. R., et al. , Blood 62:14-31 (1983)) and elements of the hemostatic system (i.e. platelets, thrombin and fibrin) are proposed as causal for tumor cell metastasis (Weiss, L., et al_, Clin. Expl. Metas. 7:127-16 (1989)).
  • Activated platelets may enhance tumor cell adhesion to the vessel wall (Honn, K. V., et al., Biochem. Phar . 34:235-241 (1985)) and induce endothelial cell retraction (Honn, K. V., et al., FASEB. J. 3:2285-2293 (1989)) while fibrin may aid tumor cells to escape the cellular immune system (Gorelik, E. , Cancer Res. 47:809-81 (1987)). Tumor cell activation of platelets and generatio of fibrin may require thrombin. Therefore, the identification and isolation of the tumor cell prothrombogenic protein(s) responsible for thrombin generation are of considerable interest.
  • tissue factor a transmembrane glycoprotein and receptor, also associated with normal tissue (Broze, G. J., et al., J. Biol. Chem. 260:10917-10920 (1985); and Buna,
  • the second, cancer procoagulant is a cysteine proteinase that directly activates factor X (Gordon, S. G. , et al., J. Clin. Invest. 67:1665-1671 (1981)).
  • the third is a protein termed platelet aggregating activity/procoagulant activity (PAA/PCA) isolated from solid tumors or from tumor cells grown in culture which lacks proteolytic activity and is dependent upon factor X for activity (Cavanaugh, P. G., et al., Thro b. Res. 37:309-326 (1985)). More recently, a factor Xa receptor has been identified on some tumor cells (Sakai, T., et al. , J. Biol. Chem. 265:9105-9113 (1990)).
  • metastatic tumors are metastatic. If the tumor is not metastatic then the tumor can be removed. If the tumor cell is metastatic then various forms of radical surgery, chemotherapy and radiation may be indicated. It is important that there be a reliable test for metastatic tumor cells.
  • Figure la shows the results of Q-Sepharose column chroma ography of material from human ovarian tumors which were metastatic.
  • the plasma membrane fraction containing 0.25% CHAPS (3-cholamidopropyldimethyl-amminio-l-propane sulfonate) was applied onto a 1 cm x 30 cm column equilibrated in 25 mM Tris-HCl buffer, pH 7.5, containing 0.25% CHAPS.
  • the column was washed with two column volumes of starting buffer to remove unbound protein and developed with a 0.0-1.0 M NaCl gradient in 120 ml at a flow rate of 60 ml/hr.
  • the eluate was fractionated into 5 ml volumes. Absorbance at 280 n is shown as open circles.
  • Procoagulant activity is shown as closed circles. As shown in Figure la, the activity eluted in a symmetric peak. The active fractions were pooled, dialyzed against 10 mM KH2PO4/K2HPO4 buffer, pH 7.2 and applied onto a hydroxyapatite column (Source: Bio-Rad, Rockville, NY).
  • Figure lb shows the Mono Q column chromatograph
  • the column was equilibrated in 25 M Tris-HCl buffer, pH 8.2. Protein isolated from the hydroxyapatite column was delipidated. There was therefore no further requirement for detergents in this and subsequent steps.
  • the column was washed with two column volumes to remove unbound protein and developed with a 0.0-1.0 M NaCl gradient in 120 ml at a flow rate of 60 ml/hr.
  • the eluate was fractionated into 5 ml volumes. Most activity ( ⁇ 75%) was seen in fraction 18 (shaded area) . It was obvious that the activity resided in the ascending portion of another major protein peak. Some activity was also present in fractions 19 through 23 but they were not included, thus reducing contaminating proteins.
  • Figure lc shows the slab gel electrophoretic pattern of the procoagulant protein preparation after the Mono Q column chromatography of Figure lb in the presence of sodium dodecyl sulfate (lane a) and beta-mercaptoethanol (lane b) .
  • Slab gel electrophoresis with SDS was carried out according to Laemmli (Laemmli, U. K. , Nature (London). 227:680-685 (1970)). Stacking and separating gels were 4% and 9% acrylamide, respectively. Samples were diluted 1:2 in the upper gel buffer containing 2% (w/v) SDS and heated to 100°C for 5 minutes.
  • Figure 3 shows a time course of thrombin generation in normal human plasma (3A) and plasma deficient in Factor VII (3B) in the presence (closed symbols) and absence (open symbols) of HLA-DR.
  • the concentration of HLA-DR was 115 nM (Read, S. M., et al., Anal. Biochem.
  • the present invention relates to a method for testing cells for the presence of human tumor cells which are metastatic which comprises testing a sample of human cells, suspected of containing human tumor cells which are metastatic to determine the presence of Human Leukocyte Antigen (HLA), wherein the tumor cells exhibit higher HLA activity as compared to HLA activity in normal human cells.
  • the cells are preferably from human blood serum.
  • the present invention relates to a method for testing cells for the presence of human tumor cells which are metastatic which comprises testing cells of human tissue suspected of containing human tumor cells which are metastatic with a protein which selectively binds Human Leukocyte Antigen (HLA) to determine the presence of the tumor cells, wherein the tumor cells exhibit higher HLA activity as compared to HLA activity in normal human cells as determined by the binding of the HLA with the protein.
  • HLA Human Leukocyte Antigen
  • the proteins which are preferred are toxins which bind HLA.
  • the toxins are selected from the group consisting of Staphylococcus aureus endotoxin A, B, Cl, C2, C3, D and E, Toxic Shock Syndrome bacterial toxin,
  • Streptococcus pyrogenes A and C Staphylococcus aureus exfoliating toxins A and B as well as active subunits of these toxins which selectively bind the HLA.
  • Preferred is S_. aureus toxin A (SEA).
  • SEA S_. aureus toxin A
  • HLA preferably between about 1 and 10 arbitrary units (AU) per unit of HLA.
  • AU arbitrary units
  • a solution containing between about 1 and 10 AU/ml of the toxin is used to inhibit the HLA.
  • the toxins preferably selectively bind HLA-DR.
  • the test can be in a blood coagulation assay, where the HLA is inhibited by the toxin; in a titration assay using the toxin which is metered into a sample of the HLA with an end point indicator; in an immunoassay with an antibody which is specific for HLA using various known techniques. All of these types of assays are well known t those skilled in the art.
  • blood plasma is freed of fibrinogen by forming a fibrin clot which is separated from the plasma.
  • the inhibition of HLA generate thrombin in the plasma is then detected using varying amounts of the toxin added to the tumor cells or to the HL isolated from the cells. Larger amounts of HLA in metastatic cells requires larger amounts of toxin to slow the rate of thrombin generation.
  • the thrombin is detected using a colorimetric assay.
  • the inhibition of HLA induced coagulation by the toxin can also be tested as a function of the clotting time using the tumor cells with or without the toxin.
  • Such types of assays are well known to those skilled in the art.
  • a control is run with normal plasma as well.
  • the samples can be isolated from the solid tumors of various types including ovarian carcinoma cells, melanomas and small cell carcinoma of the lung. If the tumor cells show excess levels of HLA over the normal cells, they are determined to be metastatic.
  • MHO Histocompatibility Complex
  • the Class I proteins are known as the transplantation antigens while the Class II proteins are referred to as the immune response proteins (Kappes, D, et al., Ann. Rev. Biochem. 57:991-1028 (1988)).
  • Structural studies have revealed that each class of protein exists as an alpha-beta heterodimer on the surface of a variety of cell types including leukocytes, lymphocytes, macrophages and monocytes .
  • the alpha and beta chains of class II heterodimer have molecular weights of 33,000 and 28,000, respectively (Lee, J.
  • the alpha chain shares 35% homology with the beta chain (Lee, J. S., et al. , Nature. 299:750-752 (1982)), thus it is believed that these areas of sequence homology contain the peptides with the procoagulant activity.
  • Both the alpha and beta chains span the membrane. Each chain consists of two main extracellular domains of 90-100 amino acids, a transmembrane egion of 20-25 amino acids and an intracellular segment of 8-15 amino acid residues.
  • the procoagulant activity is present in intact tumor cells (Chelladurai , M. , et al . , Proc. Am. Ass. Can. Res. 29:68a (1988)) suggesting that the procoagulant activity resides with the extracellular moiety of the HLA-DR molecule.
  • DR Class II antigens
  • SPECIFIC DESCRIPTION MATERIALS AND METHODS Human ovarian carcinoma was selected since ovarian tumors have high levels of procoagulant activity and adequate amounts of starting material were available.
  • the procoagulant protein finally isolated had properties different from those ascribed to the other tumor procoagulants .
  • the protein isolated was the DR subset of the Class II major histocompatibility antigens (MHO.
  • Example 1 Human ovarian tumors were provided by The National Disease Resource Interchange, Philadelphia, Pennsylvania and The Cooperative Human Tissue Network,
  • PMSF ImM Phenylmethylsulfonyl fluoride
  • the cell homogenate was passed through a muslin gauze and the filtrate centrifuged at 6700 x g for 7 minutes in a Beckman Ti45 rotor.
  • the pellet containing the nuclear and mitochondrial fraction was resuspended in the MES buffer and the centrifugation repeated at 6700 x g for 7 minutes.
  • the supernatants were pooled and recentrifuged at 15,100 x g for 19 minutes in the same rotor.
  • the resulting pellet, the light mitochondrial fraction was resuspended in 25 mM Tris-HCl buffer, pH 8.2. CHAPS was added to the suspension to obtain a final concentration of 0.25%. Isolation of Procoagulant Protein.
  • the light mitochondrial fraction i.e.
  • Plasma membrane plus lysosomal was applied onto a 1 cm x 30 cm Q-Sepharose (Pharmacia, Piscataway, NJ) column equilibrated in 25 mM Tris-HCl buffer, pH 7.5, containing 0.25% CHAPS.
  • the column was developed as detailed in reference to Figure la.
  • Activity was quantitated in a newly developed coagulation assay described below. Active fractions were pooled, dialyzed against 10 mM KH2PO4/K2HPO4 buffer pH 7.2 containing 0.25% CHAPS and applied onto a hydroxyapatite (Bio-Rad, Rockville, NY) column equilibrated in the same buffer.
  • Procoagulant activity which eluted with the unbound fraction was dialyzed extensively against distilled water to remove CHAPS and lyophilized.
  • the dried powder was resuspended in a minimal volume of 25 mM Tris-HCl buffer, pH 8.2 and lipid extracted from the preparation by vortexing each ml of recon tituted protein sample with 0.7 ml of LipiFreeTM (1, 1 ,2-Tri-chlorotriluoro thane; Genex Corporation, Gaithersburg,MD) .
  • the cloudy suspension was centrifuged at 1000 x g for 20 minutes.
  • Protein microsequence analysis was obtained by automated Edman chemistry on an Applied Biosystems gas phase sequenator, Model 470, with on line HPLC (Model 120), a Nelson Analytical chromatography Data System and a 900 A control/data system. Levels below 100 picomoles have been sequenced routinely for proteins transferred by electroblotting to polyvinyl difluoride (PVDF; eg Immobilon P, Millipore Corporation, Burlington, MA) membranes. Lectin Affinity Chromatography. This procedure was as previously described (Roitt, I., et al. , Anal. Biochem. 116:53-64 (1985)).
  • PVDF polyvinyl difluoride
  • Monoclonal antibody to HLA-DR was reconstituted in H2O, dialyzed against 0.1 M sodium bicarbonate buffer pH 8.3 containing 0.5 M NaCl (coupling buffer) and coupled to CNBr-activated Sepharose 4B suspended in the same buffer.
  • the protein isolated from the lectin column was applied to the immunoaffinity column.
  • the column was washed with at least three column volumes to remove unbound protein and bound protein was eluated with O.l M Glycine/HCl buffer, pH 2.5. Eluted fractions were dialyzed extensively against H2O and lyophilized. Protein concentration was determined according to the Bradford method (Read, S. M., et al., Anal. Biochem. 116:53-64 (1981) ).
  • Thrombin generated in these assays was measured using S 2238 (H-D- Phenylalanyl -L- pipecoyl-L-arginine-o- nitroanilide dihydrochloride) , a thrombin-specific chromogenic substrate (Sandberg, H., et al. Throm. Res. 14:113-124 (1979)).
  • S2238 H-D- Phenylalanyl -L- pipecoyl-L-arginine-o- nitroanilide dihydrochloride
  • the color generated after addition of S2238 was stopped with 50% acetic acid and absorbance measured in a Bio-Tek EL312 Microplate Reader.
  • the unit of procoagulant activity is defined as the amount of protein required to generate one unit of thrombin per min.
  • thrombin generation For a standard curve, known amounts of thrombin were used to hydrolyze S2238 and the rate of change of optical density per minute versus thrombin concentration was obtained. Rabbit thromboplastin used, under the same conditions, in controls and was found to generate 5.5 units of thrombin per minute per g protein. By comparison, immunochemically purified HLA-DR generated 435 units of thrombin per min per mg protein. This represents a 79-fold higher activity compared to thromboplastin. Anti-human tissue factor antibody did not have any effect on the procoagulant activity of our preparations of human ovarian carcinoma. The effect of HLA-DR on thrombin generation in commercially available factor-deficient plasmas (Helena Laboratories, Beaumont, Texas) were also tested.
  • the Western blot protein transfer procedure is a modification of the Bio-Rad method. All buffers were made with Milli-Q (Millipore Corporation, Burlington, MA) grade water. Proteins in a polyacrylamide gel slab were electrically transferred (35 V constant for 16 h) onto an Immobilon P membrane (Millipore) using a Bio-Rad Trans-Blot apparatus and 25 mM Tris, 200 mM glycine, 20% methanol, pH 8.3 as the transfer buffer. The membrane was stained for 15 min with 0.25% Coomassie blue in 50% methanol. The membrane was then destained in 90% methanol. RESULTS
  • procoagulant activity was attempted using conventional methods.
  • a plasma membrane enriched fraction was isolated since it was observed that (i) intact tumor cells were able to significantly reduce clotting time in recalcified plasma and (ii) in subcellular fractionation studies the plasma membrane of the human ovarian carcinoma contained more than 85% of the total procoagulant activity (Chelladurai, M., et al., Proc. Am. Ass. Can. Res. 29:68a (1988)).
  • procoagulant activity was increased as much as 20,000 fold greater in murine tumor tissues compared to normal cells (Chelladurai, M. , et al., Proc. Am. Ass. Can. Res. 29:68a (1988)).
  • the detergent CHAPS was employed to solubilize the light mitochondrial fraction and in buffers for the first ion-exchange chromatography and hydroxyapati e column. Subsequently, all procedures were carried out in the absence of detergent. In order to reduce interference from lipids in the membrane fraction, the material containing procoagulant activity was treated with Lipifree 1 *. Following the isolation protocol described in the legends to Figures la and lb, the protein yields were poor and homogeneity was not achieved. Therefore, the semipurified protein preparation was separated on SDS-PAGE, transferred onto an immobilon P membrane and Coomassie stained to identify the protein bands (Figure lc) . Two protein bands of 35,000 and 28,000 daltons were found to have procoagulant activity. The corresponding stained bands were then excised and used for sequence analysis. The sequence of the first 12 residues of the 35,000 protein band is shown in Figure 2. A computer search matched this sequence exactly with the sequence of the major histocompatibility antigen HLA-DR.
  • HLA-DR human immunoglobulin Diffraction-DR
  • HLA-DR was isolated by lentil lectin affinity chromatography followed by immunoaffinity chromatography.
  • the protein that bound to the anti-HLA-DR monoclonal antibody column had strong procoagulant activity with a specific activity of 435 units/mg protein. This activity was about 79-fold higher than that for rabbit thromboplastin under the same conditions.
  • the procoagulant nature of HLA-DR was verified when authentic HLA-DR expressed the same procoagulant activity reported in this work.
  • HLA-DR Immunochemically isolated HLA-DR enhanced thrombin generation approximately 20-fold in normal plasma during the first four minutes of incubation of the protein in recalcified plasma (Fig. 3a) .
  • the final concentration of HLA-DR in the assay was 115 nM.
  • Thrombin generation was enhanced two-fold with 11.5 nM. Since most coagulation factors are present in plasma- at micromolar concentrations or greater (Shapiro, S. S., et al., In Hemostasis and Thrombosis (eds. E.J.W. Bowie and A.A. Sharp),
  • HLA-DR appears to be quite efficient in promoting thrombin generation.
  • the procoagulant activity of HLA-DR was completely inhibited when preincubated with equimolar concentrations of staphylococcal enterotoxin A (SEA).
  • SEA specifically binds to HLA-DR (Fleischer, B. , et al.. Cell. Immunol. 120:92-101 (1989)). This inhibition served as the basis for qualifying HLA.
  • Table 1 shows the procoagulant activity of various cell lines in a coagulation assay with plasma free of fibrinogen. The thrombin generated was tested with
  • the same cell lines were tested with SEA and the results are shown in Table 2.
  • the cell lines are various metastatic cell lines.
  • WM 1158 and WM 323 are melanoma cells.
  • Table 1 Effect of SEA on procoagulant Activity of various melanoma cell lines.
  • HLA-DR 1 s ability to generate thrombin however was dependent on all coagulant factors of the intrinsic pathway. No activity was observed with individual plasmas deficient in factors XII, XI, X, IX, VIII, or V. It is not clear at this time what mechanism(s) is involved in thrombin generation. These results on the coagulation factor requirement have to be viewed with caution, since congenitally deficient plasma known to contain no biological activity may have immunochemically detectable proteins .

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Abstract

Procédé de dépistage de cellules tumorales métastatiques se fondant sur la présence de l'antigène leucocytaire humain (HLA). On utilise lors du test une protéine qui se lie sélectivement au HLA pour déterminer la présence de HLA excédentaire, ce qui constitue une caractéristique des cellules tumorales. La protéine préférée est une entérotoxine bactérienne, de préférence l'entérotoxine A (SEA) produite par le staphylocoque doré.
PCT/US1992/006237 1991-07-29 1992-07-27 Procede de depistage de cellules tumorales metastatiques WO1993003378A1 (fr)

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US737,431 1991-07-29
US07/737,431 US5378607A (en) 1991-07-29 1991-07-29 Method for testing for the presence of metastatic tumor cells

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0552342A4 (fr) * 1991-07-29 1994-01-19 Radiation Oncology Center Research And Development Corporation
WO1998019167A3 (fr) * 1996-10-29 1998-09-03 Hutchinson Fred Cancer Res Gene de classe i du complexe majeur d'histocompatibilite humain a stress cellulaire regule
US6451734B1 (en) 1996-11-04 2002-09-17 Basf Aktiengesellschaft Substituted 3-benzylpyrazoles and their use as herbicides

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6075119A (en) 1997-04-07 2000-06-13 The Rockefeller University Peptides useful for reducing symptoms of toxic shock syndrome

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5529934A (en) * 1991-07-29 1996-06-25 Biomide Investment Limited Partnership Method for measuring blood procoagulant activity of human leukocyte antigens

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Biological Abstracts, Volume 89, No. 7, issued 01 April 1990, J.P. JOHNSON et al., "Functional aspects of three molecules associated with metastasis development in human malignant melanoma", Invasion Metastasis, 9(6), pp. 388-350, 1989 see the entire Abstract. *
International Conference on Biological Treatment of Melanoma and Other Cancers (Meeting Abstract), issued 1990, G. RIETHMULLER et al., "Cellular adhesion molecules on primary melanoma and characteristics of micrometastatic cells in various epithelial cancers", page 24, see the entire Abstract. *
See also references of EP0555436A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0552342A4 (fr) * 1991-07-29 1994-01-19 Radiation Oncology Center Research And Development Corporation
WO1998019167A3 (fr) * 1996-10-29 1998-09-03 Hutchinson Fred Cancer Res Gene de classe i du complexe majeur d'histocompatibilite humain a stress cellulaire regule
US6451734B1 (en) 1996-11-04 2002-09-17 Basf Aktiengesellschaft Substituted 3-benzylpyrazoles and their use as herbicides

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AU3350993A (en) 1993-03-02
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US5378607A (en) 1995-01-03

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